The invention relates to a thermoresponsive substrate for receiving biological cells, in particular a substrate whose surface properties are variable as a function of the temperature. Furthermore, the invention relates to a method for the preparation of such a substrate, in particular a method for the application of thermoresponsive polymer material to a substrate body. Furthermore, the invention relates to a method for the cultivation of biological cells on a thermoresponsive substrate. Applications of the invention are given in the in vitro cultivation of biological cells.
It is generally known to cultivate living biological cells on substrates external to an organism (in vitro cultivation). A substrate intended for cultivation (cultivation substrate) typically features a solid substrate body, e.g. made of glass or plastic, whose surface (carrier area) is functionalized. By means of the functionalization, comprising, for example, a plasma treatment, a coating with proteins (such as e.g. fibronectin, collagen) or a coating with polymers (such as e.g. polylysine), an interaction of the cells with the surface is influenced. There is interest in cultivation substrates allowing for targeted manipulation of the biological cells and in particular influencing of, for example, the adhesion, migration, proliferation, differentiation or cell transformation (formation of tumor cells). The gentle detachment of the cells from the surfaces in particular is a fundamental problem. This occurs typically via an enzyme treatment (trypsination) which can lead to damages to and losses of the cells.
It was found in experiments that properties of biological cells can be influenced through the hardness of the surface of the cultivation substrate. Hardness variations obtained by sequential coatings with polyacrylamide and biomolecules, for example, led to different differentiations of mesenchymal stem cells (see Discher et al. in “Cell” 126 (2006), 677-689). Furthermore, it is known that the adhesion of biological cells depends on the hardness of the substrate surface.
Furthermore, thermoresponsive polymers are known. A thermoresponsive polymer is characterized by having a switching temperature (“lower critical solution temperature” (LCST)) in aqueous media. Aqueous media are e.g. pure water, commercially available buffer solutions, cell culture media or mixtures of water with organic solvents. Below the switching temperature, aqueous solutions of thermoresponsive polymers are monophasic, above this temperature biphasic. When thermoresponsive polymers are immobilized on surfaces, they perform a phase transition (conformational transition) in aqueous media when the switching temperature is exceeded; they are more strongly hydrated below the switching temperature than above.
It was found that the adhesion on substrates which are coated with the thermoresponsive (thermosensitive) polymer poly-(N-isopropyl acrylamide) (“PNIPam”) or derivatives thereof and feature the temperature-dependent hydration can be influenced in a targeted manner as a function of the temperature (see N. Yamada et al. in “Makromol. Chem.” 11 (1990), 571; C. Williams et al. in “Adv. Mater.” 21 (2009), 2161-2164, O. Ernst et al. in “Lab Chip” 7 (2007), 1322). This property was also shown with polyethylene glycol (PEG)-based polymers (see E. Wischerhoff et al. in “Angew. Chem.” (2008), 5666).
Conventional substrates whose surfaces are coated with thermoresponsive polymers (e.g. WO 2004/011669) can have disadvantages both in terms of the preparation of the coating and the suitability for cell cultivation. For example, the preparation of a substrate coated with a thermoresponsive polymer requires several elaborate process steps which are realized with an expensive apparatus assembly. Furthermore, there is only limited variability of the polymer composition. For example, the thermal response behavior of the thermoresponsive polymer can change or disappear if a second polymer component is added to the polymer. Thus, there is only limited flexibility with regard to the introduction of another functionalization of a substrate coated with a thermoresponsive polymer.
Different protocols for the functionalization of the substrate body were developed for the preparation of substrates coated with thermoresponsive polymers, such as e.g. reactions with silanes, a plasma treatment or a chemical treatment. In this connection, functional groups, such as e.g. —NH2, —COOH or epoxides, are provided on the surface of the substrate body which enable the complementary functionalized molecules, such as, in particular, the thermoresponsive polymers, a covalent attachment. In this connection, a limited reproducibility and controllability of the functionalization, in particular with regard to the attachment density and homogeneity, as well as the limitation to specific substrate materials and chemical substances and a limitation to hard, planar substrate bodies have proven to be disadvantageous. The preparation of a surface with defined mixtures of different molecules is only possible in specific exceptional cases and with a great deal of effort.
The following property of thermoresponsive polymers has particularly proven to be disadvantageous for the cultivation of biological cells. A thermoresponsive polymer is in general a polymer which experiences a physical phase transition as a function of the temperature, wherein a rearrangement of polymer chains takes place, for example. While the phase transition in a liquid solution within a temperature range of a few ° C. is sharply defined, thermoresponsive polymers immobilized in layers are characterized by a wide temperature profile of the phase transition. Thus, it was found that a cooling-down from 37° C. to temperatures below 20° C. for up to an hour is required for certain types of adherent cells to release the adhesion from the surface of the substrate (see “Application Notes” for the PNIPam-coated UpCell cultivation substrates from the manufacturer Nunc). However, such a cooling-down for this time is undesired due to the possible influencing of the cell function associated therewith. Furthermore, it was discovered in practice that thermoresponsive polymer layers can be insufficiently effective for different cell lines, such as e.g. MCF7 tumor cells or MG63 osteoblast cells.
Conventional techniques are further characterized by disadvantages in the cultivation with so-called co-cultures. As a cell type to be cultivated requires messengers (paracrine factors) from other cells for the growth or the maintenance of vitality in the adherent state, the cultivation, the growth or manipulative or analytical processes of adherent cells often have to be performed together in the co-culture (e.g. stem cells and feeder cells or melanocytes and keratinocytes). For the subsequent separation of the cells, only methods based on a cell separation in liquid cell suspensions are available up to now. For this, the cells have to be detached from the substrate and transferred into a separating device (flow cytometer) which has significant disadvantages due to the time and preparation expenditure and the low yield. In particular for samples with cell counts of less than 105 cells, the conventional cell separation is not workable as an excessively high number of cells is lost during the formation of the suspension and the separation in the flow cytometer.